Functional characterization of α-Gal producing lactic acid bacteria with potential probiotic properties

The possibility of exploiting the human immune response to glycan α-Gal for the control of multiple infectious diseases has been the objective of recent investigations. In this field of research, the strain of Escherichia coli O86:B7 has been at the forefront, but this Gram-negative microorganism presents a safety concern and therefore cannot be considered as a probiotic. To address this challenge, this study explored the identification of novel lactic acid bacteria with a safe history of use, producing α-Gal and having probiotic potential. The lactic acid bacteria were isolated from different traditionally fermented foods (kununn-zaki, kindirmo, and pulque) and were screened for the production of α-Gal and some specific probiotic potential indicators. The results showed that Ten (10) out of forty (40) [25%] of the tested lactic acid bacteria (LAB) produced α-Gal and were identified as Limosilactobacillus fermentum, Levilactobacillus brevis, Agrilactobacillus composti, Lacticaseibacillus paracasei, Leuconostoc mesenteroides and Weissella confusa. Four (4) LAB strains with highest levels of α-Gal were further selected for in vivo study using a mouse model (α1,3GT KO mice) to elucidate the immunological response to α-Gal. The level of anti-α-Gal IgG observed were not significant while the level of anti-α-Gal IgM was lower in comparison to the level elicited by E. coli O86:B7. We concluded that the lactic acid bacteria in this study producing α-Gal have potential probiotic capacity and can be further explored in α-Gal-focused research for both the prevention and treatment of various infectious diseases and probiotic development.


Results
Lactic acid bacteria produce α-Gal. Out of the 40 LAB isolated from kununn-zaki, kindirmo, and pulque, 10 were positive for α-Gal with the isolates having a varying concentration of produced α-Gal (Fig. 1).
Taxonomic classification of α-Gal producing lactic acid bacteria. Isolates were selected for molecular identification based on their ability to produce α-Gal using 16S rRNA gene sequencing. The resulting nucleotides were BLAST searched on National Centre of Biotechnology Information (NCBI; https:// www. ncbi. nlm. nih. gov) and EZBioCLoud (https:// www. ezbio cloud. net) databases and identified as belonging to Weissella (17%) and Limosilactobacillus (83%) genera with a percentage similarity of 99-100% (Table 1). The molecular relationship between the isolates is illustrated by a phylogenetic tree (Fig. 2).
Lactic acid bacteria producing α-Gal exhibited potential probiotic and safety properties. The probiotic potential of the α-Gal producing LAB was assessed. Their tolerance to pH 2 varied among the isolates with a growth rate per hour ranging from 5.30 ± 0.02 to 15.24 ± 0.01% with isolate YA03 having the least tolerance to the acidic medium while KZ06 had the highest tolerance (Table 2). Aside Weissella confusa YA03, all the other isolates identified as Limosilactobacillus fermentum exhibited reasonable viability at pH 2 with OD between 0.8 and 0.9 like observation by Orike et al. 45 . In consideration of their tolerance to bile salt, KB30 had the best tolerability while YA03 had the lowest with a growth rate per hour in medium containing 0.3% bile that ranged from 4.52 ± 0.02 to 13.98 ± 0.01% (Table 2). All the isolates showed no haemolytic property (γ haemolysis) on   Phylogenetic tree of lactic acid bacteria producing α-Gal based on the alignment of the partial 16S rRNA sequences of the LAB isolates from this study. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.95913905 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. All positions containing gaps and missing data were eliminated. E. coli ATCC_117775T was used as an out group while LAB producing α-Gal was annotated with pink and blue colored bullets. Evolutionary analyses were conducted using MEGA6. www.nature.com/scientificreports/ Vancomycin and susceptibility to Ampicillin, Cephalothin, Chloramphenicol, Gentamicin, Oxacillin, Erythromycin and Clindamycin showed their phenotypic resistance pattern as an indicator of their safety property as regard to absence of antibiotic resistance (Table 3). Although, isolate KA20 had an intermediate susceptibility to erythromycin, in general none of the selected LAB had an antibiotic resistance profile that is of safety concern. Further, in exploring their ability to adhere to epithelial cells of the gastrointestinal tract which is an important property to determine their propensity to attach to the GIT, their cell surface hydrophobicity (CSH) was studied. All tested strain had > 65% hydrophobicity ability irrespective of the hydrocarbon used with the value ranging from 65.21 ± 0.79-83.12 ± 0.83% in xylene and 65.45 ± 1.22-84.50 ± 1.15% in chloroform ( Fig. 3) L. fermentum KA20 had the highest CSH which was 83% while L. fermentum KB30 had the least with 68% which is consistent with the findings by Harnentis et al. 46 .
Selected lactic acid bacteria did not increase the level of anti α-Gal antibodies in α-Gal deficient mouse. Previous studies reported that oral administration with bacteria that produce α-Gal in its membrane modified anti α-Gal antibodies response 17,31 . To check the effect of lactic acid bacteria, α1,3GT KO mice were fed with the selected lactic acid bacteria, L. brevis (LBH1073), L. composti (LBH1074), L. paracasei (LBH1066), and L. mesenteroides (LBH1148), and a positive control with E. coli O86:B7 by oral administration. Sera samples were collected during the experiment and anti-α-Gal antibodies were measured by ELISA. Oral administration with E. coli O86:B7 induced an anti-α-Gal IgM response overtime, and a positive tendency to increase on anti-α-Gal IgG was observed. However, anti-α-Gal IgM response observed in mice which received the oral administration of the selected lactic acid bacteria producing α-Gal were much lower at day 27 as compared to E. coli O86:B7. The anti-α-Gal IgM titers decreased while an increase was observed with positive control, an indication of an opposite effect of the LAB strains to that of E. coli O86:B7. However, there was no effect in IgG across all tested isolates including positive control as the levels of anti-α-Gal IgG were not significant between days 0 and 27 (Fig. 4). Although anti-α-Gal IgE antibodies are not associated with protective response, future studies should include these antibodies as an indicator of possible allergic reactions to α-Gal.

Discussion
Isolate YA03 was identified as Weissella confusa but due to its low tolerance of acidic medium and bile, it was discarded from our experiments. Furthermore, this species has been implicated in opportunistic infections 47 even though it has been studied and suggested as a probiotic organism due to its natural presence in traditional fermented foods 25,48,49 . The remaining isolates were identified as L. fermentum, L. brevis, L. composti, L. paracasei,     www.nature.com/scientificreports/ Leuconostoc mesenteroides which according to several studies is a predominant LAB associated with traditional fermented food 26,30 . One of the key properties considered when selecting a probiotic is its ability to thrive under acidic conditions and be able to tolerate bile salt concentration in the human gut 45,50 . The test for the probiotic potential is extensive, but preliminary investigation in this study reveals that, apart from isolate YA03 which had the lowest tolerance to pH 2 and 0.3% bile, all other isolates were able to tolerate the medium with some level of variability. The variability is expected as the ability to tolerate the harsh condition in the gastrointestinal tract is strain dependent. Considering their growth rate, isolate KZ06 was the most tolerant to acidic medium, while isolate KB30 had better growth in bile medium. Nevertheless, 83% of the α-Gal producing LAB had good tolerance to the acidic and bile medium and were considered to have the probiotic potential for further analysis. This result agrees with the findings of Obioha et al. 26 who observed no major growth inhibition of L. fermentum from dairy product in Enugu for a period of 3 h that it was monitored in acidic medium.
Another desirable property of a probiotic is its ability to colonize and adhere to the intestinal wall such that they are not easily detached during bowel movement. All isolates exhibited hydrophobicity greater than 65% to hydrocarbon. There was no observable statistical difference (p < 0.05) in their ability to adhere to either xylene or chloroform. The result obtained in our study is like those reported previously that selected L. fermentum as a potential probiotic among all isolates studied for having the highest hydrophobicity 51,52 . The ability to adhere to the mucosal layer is made possible by cell surface proteins and especially mucous adhesion-promoting proteins 52 .
In the selection of a probiotic, one of the mandatory properties is the absence of hemolytic activity, a virulence factor common to pathogenic microorganisms. None of the strains showed neither α nor β hemolytic property, a result consistent with previous reports 28, 53 .
In addition to safety concerns, LAB should normally be susceptible to erythromycin and clindamycin, but when resistance to erythromycin is observed it has been linked to erythromycin resistance operon-ermB, ermC, ermT, ermG. As for clindamycin, resistance to it has not been genetically understood and whenever it is observed there is usually the presence of erythromycin resistance operon. In many reports, LAB has been shown to be susceptible to erythromycin while they have natural resistance ability to vancomycin 41 . The importance of antibiotic resistance in probiotic selection is to determine if resistance is intrinsic or extrinsic. When resistance to vancomycin is observed, it is intrinsic but when there is resistance to erythromycin or clindamycin it is an acquired resistance and the danger lies in its transferability 54 . In this study aside from isolate KA20 that showed intermediate susceptibility to clindamycin, all other isolates can be considered safe without any potential of carrying transferable antibiotic resistance genes.
The bacterial isolates from this study that are positive for α-Gal production and showed potential probiotic properties need further studies for their possible usage in developing a functional food for immune modulation against some infectious diseases 29,55,56 . Indeed, they can be considered for vaccine development, as carbohydrate-based vaccines are good considerations in control of some pathogens 17 and can also be considered as a single-antigen pan-vaccine for the control of multiple infectious diseases 57 . Taking into consideration that it is an evolving area of research, a new humanized mouse model has been bred that lacks α-Gal epitopes just like humans with the ability to secrete antibodies against the glycan 58 . This will bridge the gap between the animal model and humans and will be very useful in preclinical investigations. Here, an α1,3GT KO mice were used to investigate the immune response to some selected lactic acid bacteria producing α-Gal. In contrast to the reported anti-α-Gal immune response by α1,3GT KO mice to E. coli O86:B7, the anti-α-Gal IgM and IgG level observed were not significant.
Gram-positive bacteria cell wall components do not have highly immunogenic surfaces like Gram-negative bacteria, which might be a reason for the low immune response observed [11][12][13] . The structure of glycans is also dependent on the source and the primary structure varies between bacteria 59 , and so it should be further explored and compared between LAB and E. coli. Another approach could be a prolonged feeding which may change the gut microbiota composition and when α-Gal producing microbes becomes dominant the immune response might be different by raising the levels of circulating α-Gal specific IgM and IgG antibodies during feeding. Engineering a LAB strain to secret lipopolysaccharide specific peptides may be also used to increase the immunogenic surface of α-Gal producing LAB. Even though the genetic manipulation of lactobacilli is very challenging and takes many years before achieving success with strain of interest this study suggests that it is a possibility that could be explored. Likewise, a continuous feeding with the selected α-Gal producing LAB could modify the composition of the gut microbiota and if dominant might modulate the immune response contrary to what was observed in this study. As reported by Mangold et al. 42 , anti-alpha-Gal antibody titers remain unaffected by the consumption of fermented milk containing Lactobacillus casei in healthy adults and taxonomically closer probiotics such as E. coli Nissle 1917 have been proposed as effective therapies 60 . Nevertheless, the diversity and safety within LAB makes it a good source to explore for promising probiotic strains which this study suggests.
In conclusion, this study is the first attempt to isolate LAB from fermented food for the detection of α-Gal production. The results identified bacteria such as Limosilactobacillus fermentum, Levilactobacillus brevis, Agrilactobacillus composti, Lacticaseibacillus paracasei and Leuconostoc mesenteroides containing α-Gal as a potential candidate effective and safe probiotic for the prevention and treatment of infectious diseases. The low immunological response observed from in vivo study could be mitigated by engineering specific strains with modified immunogenic surface, elongated feeding time or continuous search of newer strains with better immune modulation. Phenotypic characterization of the isolates. The bacterial isolates were tested for their Gram reaction using a Gram staining kit (Hi-Media, Mumbai), cell morphology was observed at 100 × power microscope (Zeiss, Germany) and catalase reaction using 3% (v/v) hydrogen peroxide was carried out to determine the presence of catalase enzyme. Physiological properties such as growth at 15 °C and 45 °C temperature, different Sodium chloride (NaCl) concentrations (2%, 4% and 6.5% w/v), CO 2 production from glucose, growth at pH 3, 0.3% bile salt and carbohydrate fermentation profile were studied (unpublished data) 44 . . The raw data of the sequenced DNA chromatogram were checked using FinchTV. The FASTA file was exported to the MEGA software and aligned using Cluster W package. The obtained 16S rRNA nucleotide sequences of α-Gal producing LAB strain were compared with other reported LAB deposited in NCBI GenBank and BLAST search. A 97-100% similarity to GenBank database was in the same operational taxonomy unit (OUT). The evolutionary analyses were conducted in MEGA 6 by creating a phylogenetic tree of lactic acid bacteria producing α-Gal based on the alignment of the partial 16S rRNA sequences. The evolutionary history was inferred using the Neighbor-Joining method with a bootstrap value of 1000 replicates. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site.

Examination of probiotic properties of the lactic acid bacteria isolates. Tolerance to low
pH. Overnight culture of LAB isolates actively growing were harvested by centrifugation (5000 rpm, 10 min, 4 °C) and were washed twice in PBS (pH 7.2). The harvested cells were re-introduced into MRS broth medium lowered to pH 2 using 1 N hydrochloric acid (HCL) followed by incubation at 37 °C under anaerobic condition. Tolerance to low pH was assessed in triplicates by monitoring the cell growth at OD 620 every hour at 37 °C for 3 h according to the methods used by Orike et al. 45  Tolerance to 0.3% bile salt. Taking into consideration that the average bile concentration is on average of 0.3% (w/v) in the intestine and the staying time of food in the stomach is projected to be 4 h 62 , the test was carried out using these parameters. Active cultures of LAB were inoculated into MRS medium containing 0.3% bile (Hi-Media) and incubated under anaerobic conditions. Cell growth was monitored every hour and tolerance to 0.3% Bile was assessed in triplicates by taking the OD at 620 nm every hour at 37 °C for 3 h according to Yi et al. 50 methodology with slight modification. MRS broth at pH 6.5 with cell suspension and MRS broth (pH 6.5) with no cell suspension served as positive control and negative control respectively. The percentage growth rate per hour was calculated using the same formula described above.
Hemolytic activity. Overnight culture of LAB isolate was used for the hemolytic activity test in line with the method of Maheshwari et al. 53 . The actively growing LAB isolates were streaked on Columbia blood agar plates containing 5% sheep blood followed by 24 h incubation in an anaerobic jar at 37 °C. Afterwards, the plates were www.nature.com/scientificreports/ observed for hemolytic features indicated by the colour and clear zones around the bacteria colonies and designated as, γ-non-hemolytic, β and α hemolysis.
Antibiotic resistance. Overnight LAB broth culture (100 µl) was pipetted into petri plates in triplicate. Thereafter, 10 ml MRS agar (Hi-Media) was aseptically poured into the petri plate and gently swirled. It was left to solidify before antibiotic discs (Ampicillin (AMP); Cephalothin (CEP); Clindamycin (CD); Chloramphenicol I; Erythromycin I; Gentamicin (GEN); Oxacillin (OX); Vancomycin (VA) were placed on it and kept at room temperature for 30 min to allow the antibiotics to diffuse 63 . The petri dishes were placed in an incubator with 5% CO 2 for 18 h at 37 °C and the observed zones of inhibition were measured. The zone of inhibition recorded was compared to the standards to determine susceptibility and resistance based on the Minimal Quality Control recommendations for Streptococcus pneumonia ATCC 49,619 and Escherichia coli ATCC 25,922 used as standard to determine zone of inhibition equivalent to susceptibility. The zone of inhibition between 10-15 mm was considered as intermediate sensitivity, greater than 16 mm is regarded as sensitive while observation of zone of inhibition up to 10 mm was considered as resistant.
Microbial adhesion to hydrocarbons. In vitro adhesion was determined by measuring the cell surface hydrophobicity to hydrocarbons using the microbial adhesion to hydrocarbons (MAHC) principle following the methods of Yi et al. 50  Sera sampling. Mice were bled on days 0th, and 21st. The blood sampling was performed by submandibular bleeding using a lancet according to the Guide to the Ethical Evaluation of Animal Studies, 7.5% circulating mouse blood (~ 100 μl) with a recovery period at least of one week, ensuring the welfare of the mice used. For separation of serum from total blood, a sterile tube without anticoagulant was used. The blood from each mouse was maintained in standing position at room temperature (RT) for clotting (20-30 min) and centrifuged at 2000 × g for 10 min in a refrigerated centrifuge. Serum was collected and conserved at − 80 °C until used for analysis.
Determination of anti-α-Gal IgM and IgG antibody levels. Antibodies positive against α-Gal Galα1-3Gal epitope were measured in mice sera using a previously reported protocol 12  www.nature.com/scientificreports/